The safety profile of a cationic lipid-mediated cystic fibrosis gene transfer agent following repeated monthly aerosol administration to sheep.

Clinically effective gene therapy for Cystic Fibrosis has been a goal for over 20 years. A plasmid vector (pGM169) that generates persistent expression and reduced host inflammatory responses in mice has raised prospects for translation to the clinic. The UK CF Gene Therapy Consortium is currently evaluating long-term repeated delivery of pGM169 complexed with the cationic lipid GL67A in a large Multidose Trial. This regulatory-compliant evaluation of aerosol administration of nine doses of pGM169/GL67A at monthly intervals, to the sheep lung, was performed in preparation for the Multidose Trial. All sheep tolerated treatment well with no adverse effects on haematology, serum chemistry, lung function or histopathology. Acute responses were observed in relation to bronchoalveolar cellularity comprising increased neutrophils and macrophage numbers 1 day post-delivery but these increases were transient and returned to baseline. Importantly there was no cumulative inflammatory effect or lung remodelling with successive doses. Molecular analysis confirmed delivery of pGM169 DNA to the airways and pGM169-specific mRNA was detected in bronchial brushing samples at day 1 following doses 1, 5 and 9. In conclusion, nine doses of pGM169/GL67A were well tolerated with no significant evidence of toxicity that would preclude adoption of a similar strategy in CF patients.

Detection of CFTR transgene mRNA expression in respiratory epithelium isolated from the murine nasal cavity.

BACKGROUND: When assessing the efficacy of gene transfer agents (GTAs) for cystic fibrosis (CF) gene therapy, we routinely evaluate gene transfer in the mouse nose and measure transfection efficiency by assessing transgene-specific mRNA using the real-time (TaqMan) quantitative reverse transcriptase-polymerase chain reaction. TaqMan is traditionally used to quantify expression in whole tissue homogenates, which in the nose would contain many cells types, including respiratory and olfactory epithelium. Only the respiratory epithelium is a satisfactory model for human airway epithelium and therefore CFTR gene transfer should be specifically assessed in respiratory epithelial cells (RECs). METHODS: We have compared laser microdissection, pronase digestion and nasal brushing for: (i) the ability to enrich RECs from the wild-type mouse nose and (ii) the length of time to perform the procedure. Using TaqMan, we subsequently assessed gene transfer in enriched RECs after nasal perfusion of GL67A/pCF1-CFTR complexes in a CF mouse model. RESULTS: Laser microdissection successfully isolated RECs; however, time-consuming sample preparation made this technique unsuitable for high-throughput studies. Pronase digestion was sufficiently rapid but only yielded 19% (range = 13%) RECs (n = 6). The nasal brushing method was superior, yielding 92% (range = 15%) RECs (n = 8) and was equally effective in CF knockout mice (91%, range = 14%, n = 10). Importantly, gene transfer was detectable in brushed RECs from 70% of perfused mice and the number of vector-specific transcripts was comparable to 3.5% of endogenous wild-type Cftr levels. CONCLUSIONS: Isolation of RECs by brushing allows accurate assessment of GTA transfection efficiency in an experimental system that is relevant for CF gene therapy.

Optimizing aerosol gene delivery and expression in the ovine lung.

We have developed the sheep as a large animal model for optimizing cystic fibrosis gene therapy protocols. We administered aerosolized gene transfer agents (GTAs) to the ovine lung in order to test the delivery, efficacy, and safety of GTAs using a clinically relevant nebulizer. A preliminary study demonstrated GTA distribution and reporter gene expression throughout the lung after aerosol administration of plasmid DNA (pDNA):GL67 and pDNA:PEI complexes. A more comprehensive study examined the dose-response relationship for pDNA:PEI and assessed the influence of adjunct therapeutic agents. We found that the sheep model can differentiate between doses of GTA and that the anticholinergic, glycopyrrolate, enhanced transgene expression. Dose-related toxicity of GTA was reduced by aerosol administration compared to direct instillation. This large animal model will allow us to move toward clinical studies with greater confidence.